1. Field of the Invention
The present invention concerns lysine-containing proteins or peptides modified by a lysine modifying agent such as an aromatic acid anhydride compound, and arginine-containing proteins or peptides modified by an arginine modifying agent, which modified proteins or peptides are useful for preventing HIV-1 or HIV-2 infection by binding to the cellular CD4 receptors for these viruses.
2. Background Information
Human immunodeficiency viruses ("HIV") have been known as the causative virus for AIDS (Acquired Immunodeficiency Syndrome). The prevalence of AIDS cases is presently increasing at an alarming rate.
Two related retroviruses that can cause AIDS are human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2). The genomes of these two viruses are about 50% homologous at the nucleotide level, contain the same complement of genes, and appear to attack and kill the same human cells by the same mechanism.
HIV-1 was identified in 1983. Virtually all AIDS cases in the United States are associated with HIV-1 infection. HIV-2 was isolated in 1986 from West African AIDS patients.
HIV-1 and HIV-2 are retroviruses in which the genetic material is RNA, rather than DNA. The HIV-1 and HIV-2 viruses carry with them a polymerase (reverse transcriptase) that catalyzes transcription of viral RNA into double-helical DNA.
The viral DNA can exist as an unintegrated form in the infected cell or be integrated into the genome of the host cell. As presently understood, the HIV enters the T4 lymphocyte where it loses its outer envelope, releasing viral RNA and reverse transcriptase.
The reverse transcriptase catalyzes synthesis of a complementary DNA strand from the viral RNA template. The DNA helix then inserts into the host genome where it is known as the provirus. The integrated DNA may persist as a latent infection characterized by little or no production of virus or helper/inducer cell death for an indefinite period of time. When the viral DNA is transcribed and translated by the infected lymphocyte, new viral RNA and proteins are produced to form new viruses that bud from the cell membrane and infect other cells.
Attempts to treat AIDS with drugs which inhibit reverse transcriptase such as 3'-azido-3'-deoxythymidine (AZT) have not been met with a desirable degree of success. Moreover, there is a potential for toxicity with the use of anti-viral drugs. Thus there is a need for an effective and safe means to prevent and treat AIDS.
HIV infections are transmitted by means such as contaminated intravenous drug needles and through sexual contact. Sexual transmission is the most frequent (86%) route of adult HIV-1 infections worldwide (AIDS in the World, Harvard University Press, Cambridge, Mass., (1992)).
The transmission of HIV by heterosexual sex poses an especially severe problem for women. By the year 2,000, it is estimated that 90% of HIV infection will be acquired via heterosexual intercourse.
The utilization of condoms provides a substantial degree of protection against transmission of HIV infections during sexual intercourse, but a difficulty arises when condoms are not employed. Moreover, the use of condoms appears to be a culturally and socially unacceptable practice in many countries.
Although men can protect themselves from HIV infection by using condoms, women who are sexually active have no similar means. Women can encourage their male sex partners to use a condom, but may not succeed. The female condom, which is just becoming available, is expensive and there is presently no evidence that it prevents transmission of HIV.
Even maintaining a monogamous sexual relationship is no guarantee of safety, for if a woman's male partner becomes infected, he can pass the virus to her. And as more women are infected, so are more babies.
There is presently frustration in the medical field by the bleak prospect for an effective AIDS vaccine in the near future and the severe limitations of drugs that effectively and safely combat HIV.
Due to the present absence of a prophylactic anti-HIV vaccine and because of limitations of educational programs, other preventive methods have been sought. Spermicides with virucidal properties have been considered for this purpose, but their application is contraindicated by adverse effects (Bird, K. D., "The Use of Spermicide Containing Nonoxynol-9 in the Prevention of HIV Infection", AIDS, 5, 791-796 (1991)).
Anti-HIV drugs currently in use or expected to be clinically. applied in the near future (Steele, F., "AIDS Drugs Lurch Towards Market", Nature Medicine, 1, 285-286 (1995)) are mostly not targeted emergence of drug resistant mutants, and are expensive, suggesting that their application for wide use in topical chemoprophylaxis is unlikely.
Cells which are the primary targets for sexual and mucosal transmission of HIV, either in the form of free virus or virus-infected cells, have not been fully defined and may be diverse (Miller, C. J. et al., "Genital Mucosal Transmission of Simian Immunodeficiency Virus: Animal Model for Heterosexual Transmission of Human Immunodeficiency Virus", J Virol, 63, 4277-4284 (1989); Phillips, D. M. and Bourinbaiar, A. S., "Mechanism of HIV Spread from Lymphocytes to Epithelia", Virology, 186, 261-273 (1992); Phillips, D. M., Tan, X., Pearce-Pratt, R. and Zacharopoulos, V. R., "An Assay for HIV Infection of Cultured Human Cervix-derived Cells", J. Virol. Methods, 52, 1-13 (1995); Ho, J. L. et al., "Neutrophils from Human Immununodeficiency Virus (HIV)-Seronegative Donors Induce HIV Replication from HIV-infected Patients Mononuclear Cells and Cell lines": An In Vitro Model of HIV Transmission Facilitated by Chlamydia Trachomatis., J. Exp. Med., 181, 1493-1505 (1995); and Braathen, L. R. & Mork, C. in "HIV infection of Skin Langerhans Cells", In: Skin Langerhans (dendritic) cells in virus infections and AlDS (ed. Becker, Y.) 131-139 (Kluwer Academic Publishers, Boston, (1991)). Such cells include T lymphocytes, monocytes/macrophages and dendritic cells, suggesting that CD4 cell receptors are engaged in the process of virus transmission (Parr, M. B. and Parr, E. L., "Langerhans Cells and T lymphocyte Subsets in the Murine Vagina and Cervix", Biology of Reproduction, 44, 491-498 (1991); Pope, M. et al., "Conjugates of Dendritic Cells and Memory T Lymphocytes from Skin Facilitate Productive Infection With HIV-1", Cell, 78, 389-398 (1994); and Wira, C. R. and Rossoll, R. M., "Antigen-presenting Cells in the Female Reproductive Tract: Influence of Sex Hormones on Antigen Presentation in the Vagina", Immunology, 84, 505-508 (1995)).
Therefore agents blocking HIV-CD4 binding are expected to diminish or prevent virus transmission. Soluble recombinant CD4 cannot be considered for this purpose since high concentrations are required to neutralize the infectivity of primary HIV isolates (Daar, E. S., Li, X. L., Moudgil, T. and Ho, D. D., "High Concentrations of Recombinant Soluble CD4 are Required to Neutralize Primary Human Immunodeficiency Virus Type 1 Isolates", Proc. Natl. Acad. Sci. U.S.A., 87, 6574-6578 (1990)), and in the case of SIV, the infectivity is enhanced by CD4 (Werner, A., Winskowsky, G. and Kurth, R., "Soluble CD4 Enhances Simian Immunodeficiency Virus SIVagm Infection", J. Virol., 64, 6252-6256 (1990). However, anti-CD4 antibodies are expected to prevent virus transmission independently of subtype and variability, but their application would be too costly (Daar et al, supra, Watanabe, M., Boyson, J. E., Lord, C. I. and Letvin, N. L. "Chimpanzees Immunized with Recombinant Soluble CD4 Develop Anti-self CD4 Antibody Responses with Anti-human Immunodeficiency Virus Activity", Proc. Natl. Acad. Sci. USA., 89, 5103-5107 (1992); and Perno, C.-F., Baseler, M. W., Broder, S. and Yarchoan, R., "Infection of Monocytes by Human Immunodeficiency Virus Type 1 Blocked by Inhibitors of CD4-gp120 Binding, Even in the Presence of Enhancing Antibodies", J. Exp. Med., 171, 1043-1056 (1990)).
There is a need for a safe and effective substance that can be inserted into the vagina by a foam, gel, sponge or other form to prevent HIV-1 or HIV-2 from infecting cells in the body. It is hoped that such substance be used by a woman without her partner's knowledge.
Prospects for the near and possibly not so near future to prevent HIV-1 transmission by vaccination do not seem good. A recent report that vaccination with inactivated SIV did not protect African Green monkeys against infection with the homologous virus notwithstanding a strong immune response to SIV does not appear to be encouraging in this respect (Siegel, F., Kurth, R., and Norley, S., (1995), "Neither Whole Inactivated Virus Immunogen nor Passive Immunoglobulin Transfer Protects Against SIV.sub.agm Infection in the African Green Monkey Natural Host", J. AIDS, 8, 217-226) Considering this problem, emphasis has been put on attempts to build a chemical barrier to HIV-1 transmission (Taylor, (1994), "Building a Chemical Barrier to HIV-1 Transmission", J. NIH Res., 6, 26-27).
The development of topically applied microbicides, expected to prevent sexual (mucosal) transmission of HIV-1, was suggested to need to be "effective against all sexually transmitted diseases and should not be seen, smelled, or felt while in use." It should also be inexpensive and widely available, and $25 million was expected to be devoted to its development in the United States in 1995 (Taylor, (1994) supra). Detergents (nonoxynol-9) as a universal pathogen killer have been selected for clinical trials. However, not surprisingly, this compound proved to be deleterious to the host.
Targeting the chemical barrier to transmission of individual pathogens and abandoning the requirement for microbicidal activity or combining it with other approaches would perhaps facilitate the development of compounds preventing the transmission of human immunodeficiency viruses. For example, effective blockade of receptors for the viruses might accomplish this goal. This concept may be supported by the finding that immunization of chimpanzees and rhesus monkeys, respectively, with human CD4 which has several amino acid point mutations in comparison with non-human primate CD4 sequences (Fomsgaard, A., Hirsch, V. M., and Johnson, P. R., (1992), "Cloning and Sequences of Primate CD4 molecules: Diversity of the Cellular Receptor for Simian Immunodeficiency Virus/Human Immunodeficiency Virus", Eur. J. Immunol., 22, 2973-2981), developed anti-CD4 antibodies which inhibited HIV-1 and SIV replication (Watanabe, M., Levine, C. G., Shen, L., Fisher, R. A., and Letvin, N. L. (1991), "Immunization of Simian Immunodeficiency Virus-Infected Rhesus Monkeys with Soluble Human CD4 Elicits an Antiviral Response," Proc. Natl. Acad. Sci. USA, 88, 4616-4620. Watanabe, M., Chen, Z. W., Tsubota, H., Lord, C. I., Levine, C. G., and Letvin, N. L., (1991), "Soluble Human CD4 Elicits an Antibody Response in Rhesus Monkeys that Inhibits Simian Immunodeficiency Virus Replication", Proc. Natl. Acad. Sci. USA, 88, 120-124; and Watanabe, M., Boyson, J. E., Lord, C. I., and Letvin, N. L., (1992), "Chimpanzees Immunized with Recombinant Soluble CD4 Develop Anti-self CD4 Antibody Responses with Anti-human Immunodeficiency Virus Activity", Proc. Natl. Acad. Sci. USA, 89, 5103-5107).
In an effort to expand the diversity of compounds with medically useful biological activities, the chemical transformation of synthetic peptide-based or other combinatorial Libraries of organic compounds has been recently conceived (Ostresh, J. M., Husar, G. M., Blondelle, S. E., Dorner, B., Weber, P. A., and Houghten, R. A., (1994), "Libraries from Libraries: Chemical Transformation of Combinatorial Libraries to Extend the Range and Repertoire of Chemical Diversity," Proc. Natl. Acad. Sci. USA, 91, 11138-11142). Such transformations can be accomplished with reagents which alter chemical moieties of library constituents in a defined manner and high yield. The diversity of compounds of interest to medicinal chemistry can be also increased by applying the concept of chemical modification to natural products, either in the form of mixtures of compounds or in the form of isolated individual components.
Site-specific chemical modification of amino acid residues in proteins has been widely used in structure/function studies in which a loss or decrease of biological activity was related to chemical modification of specific amino acid residues. Methods for covalent chemical modification of C, M, H, K, R, W, Y residues and carboxyl groups were described and applied to many proteins (Lundblad, R. C., (1991), Chemical Reagents For Protein Modification, CRC Press, Boca Raton, Fla.). In a few cases, it was reported that changes in net electric charge caused by chemical modification of proteins increased their activity ["cationized" protein antigens were reported to have increased or altered immunogenicity (Muckerheide et al., 1987; Suzuki et al., 1992)] or mimicked changes occurring during in vivo protein turnover, causing binding of the chemically modified proteins to scavenger receptors on cells, (Westwood, M. E., McLellan, A. C., and Thornalley, P. J., (1994), "Receptor-mediated Endocytic Uptake of Methylglyoxal-modified Serum Albumin. Competition with Advanced Glycation End Product-modified Serum Albumin at the Advanced Glycation End Product Receptor", J. Biol. Chem., 269, 32293-32298; Lo, T. W. C., Westwood, M. E., McLellan, A. C., Selwood, T., and Thornalley, P. J., (1994), "Binding and Modification of Proteins by Methylglyoxal Under Physiological Conditions. A kinetic and Mechanistic Study with N.alpha.-Acetylarginine, N.alpha.-Acetylcysteine, and N.alpha.-Acetyllysine, and Bovine Serum Albumin", J. Biol. Chem., 269, 32299-32305; Abraham, R., Singh, N., Mukhopadhyay, A., Basu, S. K., Bal, V., and Rath, S., (1994), "Modulation of Immunogenicity and Antigenicity of Proteins by Maleylation to Target Scavenger Receptors on Macrophages," J. Immunol., 154, 1-8).
Heretofore U.S. Pat. Nos. 5,164,486 and U.S. Pat. No. 5,256,412 (hereinafter collectively referred to as "Tsunoo et al") disclosed an anti-HIV agent comprising a plasma protein of which the polarity of at least one amino group was chemically modified into a negatively charged moiety by using aliphatic acid anhydrides. Tsunoo et al discussed treatment, not prevention of HIV infection, preferably by intravenous administration. Tsunoo et al mentioned maleic anhydride and succinic anhydride, but did not discuss aromatic acid anhydrides. The treated proteins blocked fusion of infected cells with uninfected cells by blocking HIV-1 mediated fusion. Tsunoo et al described plasma proteins such as human serum albumin, human immunoglobulin, human transferrin and human fibrinogen, but did not discuss milk, casein or whey.
Jansen et al (WO 92/15316) and "Potent In Vitro Anti-Human Immunodeficiency Virus-1 Activity of Modified Human Serum Albumins", Molecular Pharmacology, 39, 818-823 (1991) ) described the use of cis-aconitic anhydride, propane-1,2,3-tricarboxylic acid anhydride, acetic anhydride, propionic anhydride, butyric anhydride, glutaric anhydride phthalic anhydride, and maleic anhydride to modify protein and polypeptides from proteins such as serum albumin to prepare anti-viral pharmaceuticals, by imparting a negative charge to the proteins or polypeptides. The examples in WO 92/15316 were carried out only with aliphatic acid anhydrides.
The antiviral activity of the compounds discussed in the two preceding paragraphs is considered to be ascribed to their interference with virus-induced fusion and to the possible binding of the compounds to the HIV-1 fusion domain.
However, to the best of applicants' knowledge, there have not been reported attempts to systematically modify protein amino acid residues of one or more kinds in order to (1) generate compounds with medically important biological properties, of which the original protein was totally devoid; (2) produce at the same time a compound(s) having the particular biological activity optimized and (3) generate compounds for binding to cell receptors for viruses which interfere with the initial binding of viruses to cells, thereby preventing infection.